Method of examining cell

ABSTRACT

An object of the present invention is to provide a more accurate diagnostic method of a tissue or a cell for carrying out definite diagnosis of cancer, drug resistance test and prognosis. More accurate examination of cancer tissues or cancer cells is enabled by a method of the examination of a tissue and a cell which comprises measuring at least two or more kinds of cell cycle related proteins and finding each relative value, i.e., carrying out cell cycle profiling. Furthermore, according to the profiling, to provide a tailor made therapeutic drug for cancer, and to employ a therapeutic method of cancer will be enabled in the future.

TECHNICAL FIELD

The present invention relates to a profiling method of a tissue and acell by simultaneously measuring cell cycle related proteins on multiparameters, and specifically, relates to a molecular diagnostic methodof a cancer tissue or a cancer cell.

BACKGROUND ART

Cancer cells are characterized by unlimited proliferation. If means forselectively inhibiting proliferation of cancer cells without impairingthe proliferation ability of normal cells can be found, termination oftumor growth is enabled irrespective of extent of differentiation orpenetration of the tumor, or degree of metastasis. Development ofanticancer agents targeting for proliferating cells has been extensivelycarried out to provide a therapeutic drug of cancer. However, suchanticancer agents have involved significant problems of accompanyingcell toxicity because they also inhibit the proliferation of normalcells.

In an attempt to solve such problems, it is interesting to comprehend asto how division of cancer cells differs from that of normal cells.

Cell proliferation according to a cell cycle is determined by aholoenzyme formed by a combination of a protein called cyclin, theconcentration of which varies through the cell cycle, and cyclindependent kinase (hereinafter, referred to as “CDK”) which is convertedinto its active form upon binding. Further, the holoenzyme which is acyclin/CDK complex is inhibited by a protein called cyclin dependentkinase inhibitor (hereinafter, referred to as “CDKI”) including aprotein p21 WAF1/CIP1 (p21).

Various studies have been conventionally made on proteins of cyclin Egene, D gene and B1 gene, and CDK that corresponds to each cyclindependent kinase. Cyclin/CDK2 complex and cyclin D/CDK4 or cyclin D/CDK6complex control cell cycle progress through cell cycle checkpoint duringthe G1 phase and the S phase that is a DNA synthesis phase. Also, cyclinB1/CDK1 complex controls cell cycle checkpoint immediately beforemitosis.

An invention relating to a diagnostic method and therapeutic method ofcancer focusing attention to the aforementioned cyclin/CDK complex hasbeen already disclosed in Japanese Translation Provisional PublicationNos. 2002-504683 and 2002-519681, and a method of measuring a cell cycleregulation factor without using a radioactive isotope (hereinafter,referred to as “RI”) has been also disclosed (Japanese PatentProvisional Publication No. 2002-335997).

When the state of G1 phase in a cell cycle is disrupted, abnormal cellproliferation is induced. A p53 molecule (tumor suppressing molecule)has been known as a protein playing a central role in mainly controllingthe G1 phase of a cell cycle. It has been already reported that mutationin the p53 gene is caused at a high incidence rate in a variety ofcancer tissues. A normal p53 molecule functions as a transcriptionfactor, and controls cell proliferation, DNA repair, differentiation,and apoptosis. Moreover, in noncancerous cells having a damaged DNA, thep53 molecule promotes expression of p21 cip1/kip1 that is a CDKImolecule, suppresses endogenously existing CDK2 kinase activity, andarrests the cell cycle at the restriction (R) point on the boundary ofG1/S phase. On the other hand, mutated p53 molecules can not regulatethe cell cycle, but lead to proliferation of uncontrollable cells andinstability of a genome, resulting in the cell turned into a malignanttumor.

A series of molecular pathological investigations have elucidatedclinical significance of a CDK controlling molecule in the G1 phase.Over expression of cyclin E corresponding to CDK2 that is a cell cycleregulatory protein and cyclin D1 corresponding to CDK4 and CDK6, andinactivation of CDKI molecules, e.g., p21 corresponding to CDK2, p27corresponding to CDK2, 4 and 6 and p16 corresponding to CDK2 are playingimportant roles in tumor formation in various tissues.

However, on the grounds of unestablished immunohistological techniquesdepending on specificity for each antibody and the like, ratios ofexpression of these cyclin E, cyclin D1 and the like in cancer tissuesgreatly vary with respect to each report.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a more accuratediagnostic method of a tissue and cell for carrying out definitediagnosis of cancer, drug resistance test and prognosis.

The present inventors focused attention on the presence of arelationship between existence of various types of cell cycle regulationfactors and the state of a cancer cell. As a result of elaborateinvestigations, it was found that more accurate diagnosis of cancer isenabled by measuring the expression and activity of cell cycle relatedproteins, and analyzing a cell cycle profile. Thus, the presentinvention was accomplished.

Accordingly, the present invention is directed to:

1. A method of the examination of a tissue and/or a cell which comprisesmeasuring at least two or more kinds of cell cycle related proteins, andcarrying out cell cycle profiling;

2. The method of the examination according to the above item 1 whereinthe cell cycle profiling is to obtain the information of the tissueand/or the cell by analyzing, on the basis of a measurement value of onecell cycle related protein, another relative measurement value;

3. The method of the examination according to the above item 1 whereinthe cell cycle related protein is cyclin dependent kinase;

4. The method of the examination according to the above item 2 whereinthe cell cycle related protein is at least two or more selected from anyone of CDK1, CDK2, CDK4 and CDK6;

5. The method of the examination according to the above item 2 whereinone measurement parameter of the cell cycle related protein which makesthe basis is a CDK1 activity value;

6. The method of the examination according to the above item 1 whereinthe measurement of the cell cycle related protein comprises themeasurement of expression of cyclin;

7. The method of the examination according to the above item 1 whereinthe cell cycle related protein is a cyclin dependent kinase inhibitor;

8. A method of the examination of cancer which comprises carrying outthe method of the examination of a tissue and/or a cell according to theabove item 1; and

9. A system of the examination for the method of the examinationaccording to the above item 1.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a drawing illustrating CDK kinase activities of cancer tissues(A: large intestine cancer, B: stomach cancer, C: esophagus cancer) andnormal mucosa. (Example 2)

FIGS. 2A and 2B are drawings illustrating profiling of CDK kinaseactivity. (Example 3)

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the term “cell cycle related protein” means afactor which can regulate a cell cycle, and specifically, a factor whichcan accelerate or arrest the cell cycle in a cycle starting fromdivision of a cell until the next division once again. Examples of sucha factor include e.g., cyclin, cyclin dependent kinase (CDK), as well ascyclin dependent kinase inhibitor (CDKI) and the like.

Moreover, in the present invention, the term “cell cycle profiling”means concurrent measurement of a minimum of 2 or more, preferably 3 ormore values of expression or activity of cell cycle related proteins.Preferably, the term means to obtain the information (profile) on statesor properties of a tissue and cell to be a target of the measurement byanalyzing, on the basis of a measurement value of one cell cycle relatedprotein, another relative measurement value. Examples of the measurementparameter include e.g., CDK activities such as CDK1 activity, CDK2activity, CDK4 activity and CDK6 activity; CDK expression such as CDK1protein expression, CDK2 protein expression, CDK4 protein expression andCDK6 protein expression; cyclin expression such as cyclin B proteinexpression, cyclin D protein expression and cyclin E protein expression;CDKI expression such as p16 protein expression, p21 protein expressionand p27 protein expression; p53 protein expression; Rb proteinexpression and the like.

Furthermore, the reference value for obtaining the relative value maybe, for example, a CDK1 activity value or a CDK1 protein expressionvalue, however, a measurement value other than the above parameters maybe also used.

(Measurement of Cell Cycle Related Protein)

For the measurement of the cell cycle related protein conducted in themethod of the examination of the preset invention, any measurementmethod which has been known or is going to be developed in future can beemployed. For example, the CDK enzyme activity can be measured usingconventional radioactive isotope (RI), or can be also used without usingRI (Japanese Patent Provisional Publication No. 2002-335997). In thesite of clinical examination, it is preferred that the measurement isconducted with a method in which RI is not used for readily and quicklymeasuring on a tissue collected from multiple patients or the like bybiopsy, surgical resection or the like.

Furthermore, for the measurement of the amount of protein expression,any measurement method which has been known or is going to be developedin future can be employed. For example, the measurement can be conductedwith a simple quantitative analysis system based on a dot blot techniqueby way of a protein expression analysis.

(Preparation of Sample for Cell Cycle Profiling)

For carrying out the method of the examination according to the presentinvention, preparation of a sample for the measurement is required. As aspecific method of solubilization of a cell and a method of isolation ofa sample containing CDK, those described in Japanese Patent ProvisionalPublication No. 2002-335997 may be employed.

(Analysis of Cell Cycle Profiling)

In normal cells, cell proliferation is strictly controlled by aregulation factor in the G1 phase. On the other hand, progress of thecell cycle after passing through the restriction point (R) is mildlycontrolled in comparison with the G1 phase. Therefore, it is reasonableto carry out profiling of the G1 phase CDK (CDK2, 4, 6) activity on thebasis of the G2/M kinase or CDK1 activity value.

Grounds for abnormal proliferation of cancer cells can not be simplyanalyzed with expression of a cell cycle related protein. Accordingly,in addition to alteration of the amount of expression of the protein,mutation or the like of the molecule has been reported as a cause ofinactivation or activation of these molecules. It is difficult todetermine various causes as described above with a primary assay system.Thus, it was believed that measuring the CDK activity, the regulation ofwhich involves many of cell cycle related proteins, is useful indiagnosis of cancer.

In Table 1, clinicopathological features of patients suffering fromgastrointestinal cancer determined in the Examples described below areshown. Median age of the studied group was 65 years old (in the range offrom 23 years to 86 years old). One among 22 cases of large intestinecancer, five among 8 cases of stomach cancer, and three among 7 cases ofesophagus cancer were in the clinical stage of IV phase.

Kinase activities of four kinds of CDK and expression results of ninekinds of cell cycle related proteins in normal mucosal tissues andcancer tissues derived from colon cancer patients in Table 2 and Table3, from stomach cancer patients in Table 4 and Table 5, and fromesophagus cancer patients in Table 6 and Table 7 are shown. Furthermore,results of analysis of each CDK in cancer tissues and normal mucosaltissues are shown in Table 8.

In Table 2 to Table 7, to the measurement value beyond [mean value+2SD(standard deviation)] is appended the sign of (+), while to themeasurement value less than [mean value−2SD] is appended the sign of(−). The measurement was conducted according to the process in Examplesdescribed below.

The expression of CDK was for CDK1, which exhibited a higher value inthree kinds of the cancer tissues of: large intestine, stomach andesophagus, in comparison with normal mucosa. When cut off value of theamount of CDK1 expression per total protein was determined as [meanvalue in corresponding normal tissue+2SD], the value was 1.99 ng/μg incolon, 1.60 ng/μg in stomach, and 0.91 ng/μg in esophagus.

When analysis was carried out using this cut off value, it was decidedthat the expression of CDK1 is positive in 27% of tissues of coloncancer (6/22), 75% of tissues of stomach cancer (6/8), and 57% oftissues of esophagus cancer tissue (4/7). These differences were allstatistically significant (large intestine; P<0.05, stomach; P<0.05,esophagus; P<0.05, with a Wilcoxon signed-rank test).

To the contrary, statistically significant difference was not foundbetween cancer tissues and normal tissues for the expression of CDK2, 4and 6.

Among CDK activities, marked difference was found for CDK2 betweencancer tissues and normal tissues (colon; P<0.01, stomach; P<0.01,esophagus; P<0.01, with a Wilcoxon signed-ranks test).

When cut off value of the CDK2 activity per weight of protein wasdetermined for each tissue as [mean value of the CDK2 activity exhibitedin the normal tissue+2SD], the value was 0.054 U/μg in large intestine,0.102 U/μg in stomach, and 0.033 U/μg in esophagus. Accordingly, 72% oftissues of large intestine cancer (16/22), 75% of tissues of stomachcancer (6/8), and 100% of tissues of esophagus cancer tissue (7/7) weredecided to be positive. For example, in connection with breast cancer,it was reported that expression of an isoform of cyclin E having anexcessive activity strongly correlates to survival rate of patients.This correlation suggests that high CDK2 activity is a major cause ofinstability of the genome and malignant alteration of cancer.

With respect to CDK4 activity and CDK6 activity, lower value wasobserved in cancer tissues compared to those in normal mucosa (largeintestine; CDK4 P<0.05, large intestine; CDK6 P<0.05, with a Wilcoxonsigned-ranks test).

In the stomach tissue, higher expression and activity was found for bothCDK1 and CDK2 compared to other organs. These results suggest that themucosal tissue in stomach regenerate more actively than other organs, orthickening and proliferation owing to infection with Helicobacter Pyrolioften found in Japanese may be suggested.

Technical scope of the present invention encompasses reagents andexamination systems used in methods of tissue examination and methods ofcell examination; methods of cancer examination by way of the presentmethod of the examination; and therapeutic drugs and therapeutic methodsof cancer which were selected by analyzing the profile.

Hereinafter, the present invention is explained in detail with referenceto Examples, however, the present invention is not limited thereto.

EXAMPLE 1 Method of Cell Examination

1) Preparation of Tissue Sample for Measurement

Each surgically collected tissue (2 mm³) was ground with a lysis buffercontaining Nonidet P-40 (NP-40) (manufactured by Calbiochem), andsolubilized with a homogenizer. Insoluble substances were removed by afilter. Thus resulting cell lysate was employed as a tissue sample forthe measurement.

2) Measurement of CDK Activity

A sample containing 100 μg of the total protein was added to 2 μg of anantibody (anti-CDK1, 2, 4 and 6 antibodies, manufactured by Santa Cruz)and 20 μl of protein A beads (manufactured by BioRad) to allow them toreact at 4° C. for 1 hour and to precipitate CDK molecules.

After washing with a buffer (0.1% NP-40, 50 mM Tris-HCl, pH 7.0) threetimes, 50 μl of a substrate mixture containing proteins was addedthereto followed by incubation at 37° C. for 10 minutes while shaking.

The substrate mixture contains 10 μg of histone H1 corresponding to CDK1and CDK2 (manufactured by Upstate Biotechnology, Inc.), 10 μg ofC-terminal domain of recombinant Rb protein corresponding to CDK4 and 5(a.a. 769-921), 5 mM adenosine-5′-O-(3-thiotriphosphate) (ATP-γS,manufactured by Sigma Corporation, USA), and buffer (20 mM Tris-HCl, pH7.4, 0.1% Triton X-100).

After removing the beads, monothiophosphoric acid induced into thesubstrate was incubated with a 10 mM substrate further labeled withiodoacetyl-biotin (manufactured by Pierce, USA) in a coupling buffer(100 mM Tris-HCl, pH 8.5, 1 mM EDTA) in a dark place at room temperaturefor 90 minutes, followed by termination of the reaction withβ-mercaptoethanol.

The substrate in an amount of 0.4 μg after completing the reaction wasadded onto a PVDF membrane using a slot blotter, and aspirated. Thusresulting membrane was blocked with 4% bovine serum albumin (BSA) for 30minutes, and allowed to react with avidin-FITC (manufactured by VectorLaboratories) at 37° C. for 1 hour.

After washing the membrane, images on the membrane were analyzed by afluorescent image analyzer (manufactured by BioRad). The activity wascalculated based on a standard curve corresponding to 0, 12.5, 25, 50,100 and 150 μg of CDK included in K-562 chronic myeloid leukemia cellstrain.

For reference, 1 U means an enzyme activity which is equal to that ofK-562 cell with 1 μg of total protein.

3) Measurement of Protein Expression

Surgically collected tissue (2 mm³) was ground with a lysis buffercontaining Nonidet P-40 (NP-40), and solubilized by a homogenizer.Insoluble substances were removed with a filter. Then, 2.5 μg of theprotein was added to wells (2×2×3 mm, permissible amount: 50 μl) of aslot blotter having a hydrophobic membrane (PVDF, manufactured byMillipore Corporation) set therein. Target substance in the crude samplebound on the membrane was quantitatively detected by the followingreaction, i.e., the reaction with an anti-cell cycle related proteinantibody, a biotinized secondary antibody and a fluorescent labeledstreptavidin.

During each reaction, the wells were automatically washed with TBS (25mM Tris-HCl, pH 7.4, 150 mM NaCl).

Fluorescent images on the membrane were analyzed by an image analyzer(manufactured by BioRad), and the fluorescent intensity of the dot wasmeasured. Quantitative determination of the cell cycle related proteinwas carried out with a standard curve obtained using a standardrecombinant protein (CDK1; 2.5-25 ng/dot, CDK2; 1.0-10 ng/dot, CDK4;1.0-10 ng/dot, CDK6; 2.5-25 mg/dot).

EXAMPLE 2 CDK Activity

Each enzyme activity of CDK1, 2, 4 and 6 was measured forgastrointestinal cancer tissues and normal mucosa. Measurement of theactivity was conducted with the method described in Example 1, for eachof the tissues of (A) large intestine cancer of 22 cases, (B) stomachcancer of 8 cases, and (C) esophagus cancer of 7 cases as well as normalmucosa.

The results are depicted in FIGS. 1A to C. Consequently, with respect todifference of each enzyme activity between cancer tissues and normalmucosa, statistical significance was found in elevation of the CDK2activity in the large intestine, stomach and esophagus tissues (P<0.01).On the other hand, CDK4 and CDK6 in the large intestine and stomachcancer tissues exhibited activities that were comparatively lower thanthose in normal mucosa. Statistical analysis was carried out with aWilcoxon signed-ranks test. In crease in CDK2 enzyme activity wasobserved in almost of the large intestine, stomach and esophagus cancertissues.

EXAMPLE 3 Profile of CDK activity

With respect to CDK (CDK2, 4, 6) in the G1 phase, the activity values ofCDK2, 4 and 6 were standardized on the basis of the CDK1 activity valuethat is G2/M kinase to determine the profile. Consequently, as shown bya solid line in FIG. 2A, in seven among 8 cases of normal largeintestine mucosal tissues, CDK profile in the G1 phase positioned in thefold width of 0.024 to 0.43 for CDK2/CDK1, 1.2 to 39 for CDK4/CDK1, and1.9 to 26 for CDK6/CDK1, exhibiting a common pattern. This suggests thatrelative values of the CDK activities are similar among the normal largeintestine tissues.

Next, the profile on the basis of value of CDK1 activity that is G2/Mkinase was similarly determined for CDKs (CDK2, 4, 6) in the G1 phase onrandomly selected nine cases of large intestine cancer tissue.Consequently, as shown in FIG. 2B, no profile was observed which wasidentical to the common profile found in the normal mucosal tissues

According to the present invention, it was found that more accurateexamination of cancer tissues and cancer cells is possible when cellcycle profiling that involves measurement of the activity is carriedout. It is expected that high probability to result in a prognosticfactor would be found on analyses of expression, activity, and profilingand the like of each cell cycle regulation factor in retrospective orprospective tests.

In addition, the method of the present invention in which cell cycleprofiling that involves measurement of the activity is carried out willbe extremely valuable one which can be adapted to tailor made medicaltreatments, because it was reported in connection with breast cancerthat expression of an isomer of cyclin E that imparts excessive activityto CDK2 strongly correlates to survival rate of patients.

INDUSTRIAL APPLICABILITY

When the cell cycle profiling that involves measurement of the activityis carried out according to the method of the present invention, moreaccurate examination of cancer tissues and cancer cells can be executed,and definite diagnosis of cancer, drug resistance test and prognosis areenabled. TABLE 1 Clinicopathological features of patients MetastasisLymph- Patieut Sex Age Stage Tissue-type Site node Peritoneum LiverDistant Invasion Colon cancer C001 F 53 IIIb well/mod As N2 P0 H0 M0 V1Ly1 C002 M 66 I well/mod Rs N0 P0 H0 M0 V3 Ly1 C003 M 67 IIIa well/modRs-S N1 P0 H0 M0 V1 Ly1 C005 M 56 IIIb mod-por Ac N2 P0 H0 M0 V1 Ly1C006 M 48 IIIa well-mod Rb N0 P0 H0 M0 V1 Ly1 C007 F 80 IIIa ne Rb N1 P0H0 M0 V1 Ly2 C008 M 73 II muc A N0 P0 H0 M0 V0 Ly1 C009 M 60 I well/modRs N0 P0 H0 M0 V1 Ly0 C010 M 83 IIIb well/mod T N2 P0 H0 M0 V2 Ly1 C011M 77 IIIa well/mod Rs N1 P0 H0 M0 V2 Ly1 C012 M 86 IIIa well/mod Rb N1P0 H0 M0 V2 Ly1 C013 F 71 I por > mod A N0 P0 H0 M0 V0 Ly1 C014 F 81 IIwell/mod Rs N0 P0 H0 M0 V0 Ly1 C015 F 70 III well/mod Rab N1 P0 H0 M0 V1Ly2 C016 F 43 IV well S N1 P0 H0 M1 V1 Ly1 C017 M 47 II well/mod RaRs N0P0 H0 M0 V2 Ly1 C018 M 59 II well/mod Rs N0 P0 H0 M0 V1 Ly1 C019 F 34IIIa well/mod Re N1 P0 H0 M0 V1 Ly1 C020 M 73 II well/mod A N0 P0 H0 M0V1 Ly1 C021 M 48 IIIb mod-por Ds N2 P0 H0 M0 V3 Ly3 C022 M 23 IIIb porCe N3 P0 H0 M0 V1 Ly1 C023 F 79 II well/mod Rs N0 P0 H0 M0 V1 Ly1Gastric cancer S001 M 70 IV tub2 Ue N2 P0 H1 M0 V1 Ly1 S002 M 66 IIIbpor > tub2 Ue N2 P0 H0 M0 V1 Ly1 S005 M 59 Ib tub1-2 U N0 P0 H0 M0 V2Ly2 S006 F 67 IV por2 Lm N2 P1 H0 M0 V3 Ly3 S008 M 75 IV tub2 > por1 LN2 P0 H1 M0 V3 Ly3 S009 M 67 IV tub2 M N3 P0 H1 M0 V2 Ly2 S010 M 72 IVtub2 > por Ml N2 P0 H1 M1 V2 Ly3 S011 F 40 II muc M N1 P0 H0 M0 V0 Ly2Esophageal carcinoma E002 M 46 IVa por >> mod Lt-Ae N2 M0 V1 Ly1 E003 M77 IVb mod Lt N3 M1 V2 Ly2 E005 M 44 III mod > well MtLt N3 M0 V1 Ly2E006 M 60 III mod > por UtLt N1 M0 V0 Ly2 E008 M 61 IVa mod Mt N2 M0 V2Ly2 E009 M 65 II well Mt N1 M0 V1 Ly1 E010 M 59 III mod > por Ae N3 M0V1 Ly1

TABLE 2 Each CDK activity in colon cancer patients cdk1 cdk2 cdk4 cdk6kinase kinase kinase kinase Sample name (U/ug lysate) (U/ug lysate)(U/ug lysate) (U/ug lysate) C001 0.000 0.056 (+)  0.082  0.041 C0020.049 0.072 (+) C003 0.065 (+) 0.065 (+)  0.141  0.109 C005 0.024 0.097(+) C006 0.042 0.117 (+) C007 0.020 0.057 (+) C008 0.008 0.124 (+) 0.138  0.039 C009 0.001 0.038 C010 0.000 0.179 (+) C011 0.004 0.062 (+)C012 0.024 0.034  0.020 (−) (0.032) (−) C013 0.000 0.059 (+) (0.094) (−)(0.035) (−) C014 0.037 0.068 (+) C015 0.022 0.053 (+) C016 0.009 0.051(+)  0.085  0.021 C017 0.041 0.091 (+)  0.095  0.089 C018 0.017 0.033 0.110  0.040 C019 0.000 0.045 (+)  0.099  0.154 C020 0.007 0.040(0.003) (−)  0.050 C021 0.012 0.007  0.020 (−)  0.034 C022 0.003 0.063(+)  0.012 (−)  0.012 C023 0.001 0.000  0.093  0.042

TABLE 3 Amount of each protein expression in colon cancer patient cdk1cdk2 cdk4 cdk6 cyclinD1 Sample name (ng/ug lysate) (ng/ug lysate) (ng/uglysate) (ng/ug lysate) (ng/ug lysate) C001 1.285 0.263 0.620 0.278 0.052C002 2.822 (+) 0.519 0.989 (+) 0.579 0.123 C003 1.392 0.367 0.777 0.3070.051 C005 2.606 (+) 0.492 0.970 (+) 0.405 0.089 C006 1.618 0.000 0.3430.000 0.077 C007 2.363 (+) 0.349 0.640 0.309 0.053 C008 3.022 (+) 0.3590.567 0.538 0.093 C009 0.000 (−) 0.000 0.000 (−) 0.000 0.000 (−) C0102.058 (+) 0.000 0.379 0.373 0.076 C011 1.093 0.227 0.492 0.000 0.095C012 0.673 0.164 0.348 0.000 0.046 C013 1.567 0.225 0.437 0.334 0.077C014 1.444 0.209 0.766 0.385 0.143 C015 0.869 0.000 0.192 0.000 0.074C016 1.417 0.200 0.866 (+) 0.806 (+) 0.115 C017 0.824 0.000 0.486 0.0000.058 C018 0.917 0.000 0.299 0.000 0.075 C019 0.981 0.000 0.570 0.0000.073 C020 0.800 0.194 0.149 0.000 0.041 C021 0.910 0.551 0.064 C0221.991 (+) 0.343 0.531 0.422 0.075 C023 0.800 0.202 0.517 0.000 0.069 p53p21 p16 cyclinE ELISA ELISA WB p27 Sample name (ng/ug lysate) (pg/uglysate) (mU/ug lysate) (CNT × mm) (ng/ug lysate) C001 0.501 0.134 C0020.561 0.162 0.133 C003 0.433 1.355 (+) 2.600 (−) 0.123 C005 0.762 0.1627.070 0.152 C006 0.427 1.768 (+) 5.696 0.089 C007 0.319 1.010 (+) 3.890 61.2 (+) 0.128 C008 0.691 0.513 (+) 12.690  107.5 (+) 0.114 C009 0.000(−) 0.077 (−) 4.804 0.000 (−) C010 0.303 0.054 (−) 5.607 0.117 C0110.000 (−) 0.365 (+) 1.554 (−) 0.158 C012 0.000 (−) 0.339 (+) 3.290 37.50.000 (−) C013 0.429 0.178 8.160 21.6 0.166 C014 0.371 0.067 (−) 8.0340.091 C015 0.501 0.057 (−) 1.178 (−) 0.066 C016 0.482 0.171 3.470 130.2(+) 0.107 C017 0.425 0.226 0.084 C018 1.480 0.193 4.250 35.2 0.160 C0190.618 2.963 (+) 6.970 18.2 0.089 C020 1.054 0.247 (+) 5.030 29.7 0.220C021 1.411 0.180 5.010 31.7 0.397 C022 0.468 0.224 7.890 25.4 0.128 C0230.494 0.223 12.720 19.7 0.198

TABLE 4 Each CDK activity in gastric cancer patient cdk1 cdk2 cdk4 cdk6kinase kinase kinase kinase Sample name (U/ug lysate) (U/ug lysate)(U/ug lysate) (U/ug lysate) S001 0.096 0.116 (+) (0.014) (0.038) (−)S002 0.011 0.014 S005 0.074 0.129 (+) S006 0.000 0.182 (+) 0.015  0.038(−) S008 0.027 0.181 (+) (0.012)  0.108 S009 0.000 0.555 (+) 0.067 0.024 (−) S010 0.000 0.226 (+) 0.037  0.101 S011 0.000 0.030 0.040 0.041 (−)

TABLE 5 Amount of each protein expression in gastric cancer patient cdk1cdk2 cdk4 cdk6 cyclinD1 Sample name (ng/ug lysate) (ng/ug lysate) (ng/uglysate) (ng/ugl lysate) (ng/ug lysate) S001 1.727 0.504 0.813 0.5840.105 S002 6.960 (+) 2.232 (+) 2.255 (+) 3.627 (+) 0.256 (+) S005 1.0350.000 (−) 0.282 0.000 (−) 0.051 S006 2.524 (+) 0.377 0.645 0.514 0.083S008 3.023 (+) 0.404 1.536 (+) 0.376 0.195 (+) S009 2.155 (+) 0.3300.405 0.451 0.044 S010 4.278 (+) 0.343 0.690 0.518 0.103 S011 0.7130.000 (−) 0.208 0.000 (−) 0.057 p53 p21 p16 cyclinE ELISA ELISA WB p27Sample name (ng/ug lysate) (pg/ug lysate) (mU/ug lysate) (CNT × mm)(ng/ug lysate) S001 0.518 0.158 4.050 58.0 (+) 0.141 S002 3.692 (+)0.182 8.550 0.566 (+) S005 0.300 1.279 3.824 0.109 S006 0.840 3.431 (+)8.270 78.4 (+) 0.138 S008 0.298 2.236 (+) 7.065 92.3 (+) 0.077 S0090.600 0.980 3.050 72.7 (+) 0.107 S010 0.420 0.104 16.560 (+) 68.4 (+)0.096 S011 0.312 0.000 1.150 16.7 0.086

TABLE 6 Each CDK activity in esophageal cancer patient cdk1 cdk2 cdk4cdk6 kinase kinase kinase kinase Sample name (U/ug lysate) (U/ug lysate)(U/ug lysate) (U/ug lysate) E002 0.000 0.073 (+) 0.125 0.149 E003 0.0000.034 E005 0.000 0.089 (+) E006 0.000 0.047 (+) E008 0.000 0.033 E0090.012 (+) 0.037 (+) 0.154 0.133 E010 0.002 0.048 (+) 0.015 0.201

TABLE 7 Amount of each protein expression in esophageal carcinomapatient cdk1 cdk2 cdk4 cdk6 cyclinD1 Sample name (ng/ug lysate) (ng/uglysate) (ng/ug lysate) (ng/ug lysate) (ng/ug lysate) E002 2.524 (+)0.377 (+) 0.645 0.514 (+) 0.083 E003 1.742 (+) 0.255 0.392 0.000 0.093E005 3.240 (+) 0.324 (+) 1.095 0.567 (+) 0.168 E006 0.841 0.000 0.6740.345 (+) 0.055 E008 0.484 0.000 0.000 (−) 0.000 0.000 (−) E009 0.8750.000 0.169 0.000 0.033 (−) E010 1.092 (+) 0.277 0.306 0.344 (+) 0.060p53 p21 p16 cyclinE ELISA ELISA WB p27 Sample name (ng/ug lysate) (pg/uglysate) (mU/ug lysate) (CNT × mm) (ng/ug lysate) E002 0.840 (+) 3.90.138 E003 0.391 (+) 0.099 10.749 (+) 0.081 E005 0.310 (+) 0.141  8.032(+) 0.073 E006 0.313 (+) 0.240 11.526 (+) 0.088 E008 0.000 (−) 0.1044.842 0.000 (−) E009 0.000 (−) 0.247 5.750 0.066 E010 0.000 (−) 0.126 8.560 (+) 0.083

TABLE 8 CDK1 CDK2 CDK4 CDK6 Nomal Tumor Nomal Tumor Nomal Tumor NomalTumor Activity (U/μg total protein ± s.d.) Colon 0.017 ± 0.020 0.017 ±0.019 0.012 ± 0.016 0.065 ± 0.040 0.086 ± 0.043 0.069 ± 0.051 0.094 ±0.11  0.049 ± 0.044 Stomac 0.034 ± 0.036 0.027 ± 0.038 0.040 ± 0.0310.18 ± 0.17  0.10 ± 0.086 0.027 ± 0.026  0.10 ± 0.050 0.052 ± 0.043Esophagus 0.004 ± 0.003 0.003 ± 0.004 0.011 ± 0.011 0.055 ± 0.022 0.0980.098 ± 0.073 0.16   0.16 ± 0.036 Expession (ng/μg total protein ± s.d.)Colon 0.79 ± 0.60  1.5 ± 0.78 0.23 ± 0.24 0.21 ± 0.17 0.35 ± 0.23 0.54 ±0.25 0.19 ± 0.23 0.24 ± 0.25 Stomac 0.84 ± 0.38 2.8 ± 2.0 0.39 ± 0.290.52 ± 0.71 0.38 ± 0.17 0.85 ± 0.70 0.28 ± 0.26 0.76 ± 1.2  Esophagus0.63 ± 0.14 1.7 ± 1.0 0.084 ± 0.11  0.21 ± 0.16 0.59 ± 0.39 0.55 ± 0.330.001 0.30 ± 0.25

1-9. (canceled)
 10. A method of the examination of a cell for executingdefinite diagnosis of cancer which comprises: measuring an activityvalue or amount of expression of two or more kinds of cell cycle relatedproteins in a cell collected from a patient; carrying out cell cycleprofiling on the basis of the obtained measurement value; anddetermining whether or not the cell collected from the patient is acancer cell, on the basis of the result of said profiling.
 11. In themethod of the examination according to claim 10, said profiling is ananalysis, on the basis of the measurement value of one cell cyclerelated protein, of another relative measurement value.
 12. In themethod of the examination according to claim 10, said cell cycle relatedprotein is a cyclin dependent kinase.
 13. In the method of theexamination according to claim 12, said cyclin dependent kinase is atleast two selected from the group consisting of CDK1, CDK2, CDK4 andCDK6.
 14. In the method of the examination according to claim 11, themeasurement value of the cell cycle related protein which makes thebasis is a CDK1 activity value.
 15. In the method of the examinationaccording to claim 10, said cell cycle related protein is cyclin.
 16. Inthe method of the examination according to claim 10, said cell cyclerelated protein is a cyclin dependent kinase inhibitor.
 17. A method ofthe examination of a cell for executing definite diagnosis of cancerwhich comprises: measuring an activity value or amount of expression oftwo or more kinds of cell cycle related proteins in a cell collectedfrom a patient; producing a cell cycle profile on the basis of theobtained measurement value; comparing the produced cell cycle profilewith a cell cycle profile of a normal cell; and determining whether ornot the cell collected from the patient is a cancer cell, on the basisof the result of the comparison.
 18. In the method of the examinationaccording to claim 17, said profile comprises another relative activityvalue on the basis of the activity value of one cell cycle relatedprotein.
 19. In the method of the examination according to claim 17,said cell cycle related protein is a cyclin dependent kinase.
 20. In themethod of the examination according to claim 19, said cyclin dependentkinase is at least two or more selected from the group consisting ofCDK1, CDK2, CDK4 and CDK6.
 21. In the method of the examinationaccording to claim 18, the activity value of the cell cycle relatedprotein which makes the basis is a CDK1 activity value.
 22. In themethod of the examination according to claim 17, said cell cycle relatedprotein is cyclin.
 23. In the method of the examination according toclaim 17, said cell cycle related protein is a cyclin dependent kinaseinhibitor.
 24. A method of the examination of a cell for definitediagnosis of cancer which comprises: measuring an activity value of afirst cell cycle related protein and an activity value of a second cellcycle related protein in a cell collected from a patient; producing aprofile of a relative activity value of the second cell cycle relatedprotein on the basis of the activity value of the first cell cyclerelated protein; comparing the produced profile with a profile of anormal cell; and determining whether or not the cell collected from thepatient is a cancer cell, on the basis of the result of the comparison.25. In the method of the examination according to claim 24, said firstand second cell cycle related proteins are cyclin dependent kinase. 26.In the method of the examination according to claim 25, said first cellcycle related protein is CDK1.
 27. In the method of the examinationaccording to claim 26, said second cell cycle related protein is atleast one selected from the group consisting of CDK2, CDK4 and CDK6. 28.In the method of the examination according to claim 24, said cell cyclerelated protein is cyclin.
 29. In the method of the examinationaccording to claim 24, said cell cycle related protein is a cyclindependent kinase inhibitor.